Evacuated blood collection tubes containing protease inhibitors for the assessment of contact system activation

ABSTRACT

Disclosed herein are evacuated blood collection tubes comprising protease inhibitor cocktails in liquid form and uses thereof for assessing features associated with the contact system in a subject, including the endogenous level of contact system activation, the endogenous level of a drug that targets a component of contact system during treatment, and/or the immunogenicity of such a drug.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application number PCT/US2016/046681, filed Aug. 12, 2016,which claims the benefit of the filing date of U.S. ProvisionalApplication No. 62/204,644 filed Aug. 13, 2015 and U.S. ProvisionalApplication No. 62/214,308, filed Sep. 4, 2015. The entire contents ofeach of these referenced applications are incorporated by referenceherein.

BACKGROUND OF THE INVENTION

Accurate measurement of the in vivo level of contact system activationusing patient plasma is challenging due to the propensity for ex vivoactivation during blood collection. Blood from patients with certaindiseases associated with the contact system, e.g., hereditaryangioedema, is especially prone to ex vivo contact system activationsince it is deficient in C1 inhibitor, the natural inhibitor of thepathway. Consequently, measurements of pathway specific biomarkers(e.g., 2-chain high molecular weight kininogen) may overestimate thedegree of contact system activation that is present within the patient,unless the blood is carefully collected and processed.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development ofnon-glass evacuated blood collection tubes containing protease inhibitormixtures (cocktails) in liquid formulation, which prevents ex vivoactivation of the contact system during blood collection. As such, theevacuated blood collection tubes described herein allow for accuratemeasurement of the endogenous level of contact system activation ofpatients, particularly those who are deficient in a natural inhibitor(e.g., C1 inhibitor) of this pathway.

Accordingly, one aspect of the present disclosure features an evacuatedblood collection tube, comprising a liquid formulation that comprises amixture of protease inhibitors, which may be substantially free ofprotease inhibitors that are unstable in an aqueous solution. The tubemay be a non-glass tube. In some embodiments, the tube is plastic. Insome embodiments, the evacuated blood collection tube contains 0.5 ml ofany of the liquid formulations described here, which can be diluted by10-fold in use.

In some embodiments, the mixture of protease inhibitors in the evacuatedblood collection tube described herein comprises at least one serineprotease inhibitor (e.g., a plasma kallikrein inhibitor) and at leastone cysteine protease inhibitor. In one example, the mixture of proteaseinhibitors comprises EPI-KAL2, which may be biotinylated, and leupeptin.The amount of EPI-KAL2 may range from 5 to 15 μM in the liquidformulation containing such. Alternatively or in addition, the amount ofleupeptin may range from 200 to 300 μM in the liquid formulation.

In some embodiments, the mixture of protease inhibitors described hereinmay comprise at least two serine protease inhibitors, at least one ofwhich is a trypsin inhibitor, for example, soybean trypsin inhibitor. Insome examples, the mixture of protease inhibitors comprises benzamidine,soybean trypsin inhibitor, leupeptin, and AEBSF. In some examples, theliquid formulation in the evacuated blood collection tube may comprise80-120 mM benzamidine, 1-3 mg/ml soybean trypsin inhibitor, 200-300 μMleupeptin, and/or 10-30 mM AEBSF.

The liquid formulation in any of the evacuated blood collection tubesdescribed herein may further comprise polybrene and EDTA. In someembodiments, any of the liquid formulations described herein may have apH of 4-6 (e.g., 4.5).

In another aspect, the present disclosure provides a method forassessing the endogenous level of contact system activation in asubject. The method comprises: (i) collecting blood from a subject toany of the evacuated blood collection tubes described herein; (ii)processing the blood to produce a plasma sample; and (iii) measuring thelevel of contact system activation in the plasma sample. In someembodiments, the measuring step (step (iii)) can be carried out bymeasuring the level of one or more biomarkers indicative of contactsystem activation. Such biomarkers may comprise prekallikrein, activeplasma kallikrein (pKal), α2M-pKal complex, active factor XII, activefactor XI, high molecular weight kininogen (HMWK), and/or a bradykininmetabolite. In one example, the one or more biomarkers comprise cleavedHMWK and/or intact HMWK.

In yet another aspect, the present disclosure provides a method forassessing the level of a drug targeting contact system in a subject. Themethod comprises: (i) collecting blood from a subject to an evacuatedblood collection tube as described herein, wherein the subject isadministered with a drug that targets a component of the contact system;(ii) processing the blood to produce a plasma sample; and (iii)measuring the level of the drug in the plasma sample.

Further, the present disclosure provides a method for assessingimmunogenicity of a drug targeting contact system, the methodcomprising: (i) collecting blood from a subject to an evacuated bloodcollection tube as described herein, wherein the subject is administereda drug that targets a component of the contact system; (ii) processingthe blood to produce a plasma sample; and (iii) measuring the level ofantibodies that bind to the drug in the plasma sample. Such a method mayfurther comprise, prior to step (iii), isolating antibodies that bindthe drug from the plasma sample. In some examples, the anti-drugantibodies (ADAs) can be isolated by a solid phase extraction and aciddissociation (SPEAD) assay.

In any of the methods described herein, the subject can be a humanpatient, who, in some instances, may be treated with a drug targeting acomponent (e.g., plasma kallikrein) of the contact system, for example,a drug (e.g., an antibody) specifically targeting plasma kallikrein(e.g., the active form of plasma kallikrein). In some examples, theblood is derived from a human patient having a disease associated withthe contact system, e.g., hereditary angioedema (HAE) or idiopathicangioedema. In some instances, the human patient has HAE with normalC1-inhibitor (C1-INH).

In any of the methods described herein, the evacuated blood collectiontube may not be the first tube filled with blood from the subject.Alternatively or in addition, the process step [step (ii)] can beperformed within one hour after the blood collecting step [step (i)].

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a photo showing that the SCAT169 and SCAT153 tubes preventedcontact activation as measured in a 2-Chain Western blot assay. When 10%ellagic acid is added to the plasma, the contact system is activatedleading to the conversion of 1-chain HMWK to 2-chain HMWK (see theSodium citrate plasma). In contrast, the SCAT169 and SCAT153 plasmacontains the same amount of 1-chain HMWK pre- and post-addition of theellagic acid.

FIG. 2 is a graph showing variation of kininogen cleavage (percent2-HMWK/cHMWK) based on sample collection methods in plasma from healthysubjects. The clinical site of collection, as well as the types of tubesused for collection, is indicated, including K₂EDTA (EDTA), sodiumcitrate, SCAT169, or P100. The groupings of data points correspond to,from left to right, site 1: EDTA, site 2: citrate; site 3: citrate, site4: citrate, site 1: SCAT169, site 2: SCAT169, site 4: SCAT169, site 5:SCAT169, and site 4: P100.

FIG. 3 is a graph showing kininogen cleavage (percent 2-HMWK/cHMWK) inplasma collected in SCAT 169 tubes from healthy subjects from differentclinical sites. The groupings of data points correspond to, from left toright, site 1: commercial vendor, site 4, site 5, and sites 4 and 5.

FIG. 4 is a graph showing kinongen cleavage (percent 2-HMWK/cHMWK) inhealthy subjects as compared to subjects having type I or II HAE,nC1-INH HAE, and idiopathic angioedema (AE). The groupings of datapoints correspond to, from left to right, healthy subjects, HAE I/IIbasal, HAE I/II attack, HAE (nC1-INH) basal, HAE (nC1-INH) attack,idiopathic AE basal, and idiopathic AE attack.

FIG. 5 is a graph showing 2-chain HMWK levels in plasma from healthysubjects and patients with HAE. A: shows the percentage of plasma2-chain HMWK levels from healthy subjects. As indicated in the graph,clinical site C did not use an initial discard tube before collection ofSCAT169 plasma. B: shows the percentage of plasma 2-chain HMWK levelsfrom patients with HAE.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the development ofevacuated blood collection tubes comprising protease inhibitor cocktailsthat prevent contact system activation. In order to accurately assessthe endogenous level of contact system activation in a patient or ahealthy volunteer, it is critical that blood is carefully collected andprocessed. One or more of the following precautions may be taken toensure accurate assessment of features associated with the contactsystem as described herein:

-   -   (i) The evacuated blood collection tubes described herein are        preferred not to be the first tube filled with blood, which can        show elevated contact system activation due to the local trauma        following vessel puncture by the needle;    -   (ii) The blood may not come into contact with glass (use plastic        tubes or catheters);    -   (iii) The blood may be processed to plasma within a short period        of time (e.g., ˜1 hour) after collection; and/or    -   (iv) The use of protease inhibitors in the collection tube may        stabilize the plasma against ex vivo contact activation which        masks accurate determination of endogenous patient status.

The advantages of the evacuated blood collection tubes described hereininclude, at least: (1) the use of evacuated non-glass (e.g., plastic)tubes for standardized and simplified blood collection; (2) the use of aliquid formulation comprising the protease inhibitor cocktails tominimize hydrolysis; (3) optional omission of protease inhibitors thatare unstable in aqueous solutions (e.g., PPACK II, also known asH-D-Phe-Phe-Arg-chloromethyl ketone); and (4) inclusion, in someembodiments, a plasma kallikrein inhibitor such as EPI-KAL2 (may bebiotinylated), which offers the ability for the tubes to contain areagent that permits detection of activated plasma kallikrein using animmunoassay. See, e.g., WO95/21601, the relevant disclosures thereof areincorporated by reference herein.

An unexpected observation from this study was that the use of theprotease inhibitor cocktail in liquid form prevented or reducedhemolyzation. When blood was collected into evacuated tubes containing alyophilized preparation of the protease inhibitors, it was largelyhemolyzed, which may interfere with certain analyte measurements.However, when blood is collected into an evacuated tube containing asolution of the same protease inhibitor mixture, it was not hemolyzed.

Consequently, measurements directed at assessing the degree of contactsystem activation in plasma samples processed from blood samplescollected in the evacuated tubes described herein provide more accurateassessment levels from patients with different diseases. Obtaining anaccurate estimate of contact activation can allow for identification ofdiseases or subsets of patients with different diseases that arepotentially mediated by this pathway and therefore amenable to treatmentwith an inhibitor of the contact system.

Further, the use of the evacuated blood collection tubes describedherein can facilitate the accurate determination of drug levels and/orimmunogenicity assessment for a therapeutic molecule directed againstactivated forms of proteins in the contact system (e.g., plasmakallikrein, FXIIa, and 2-chain kininogen). The tubes may offer a similaradvantage for therapeutic molecules targeted to activated proteinsdownstream of the contact system activation that do not require calciumfor the generation of the activated target (e.g., FXIa, and FIXa). Theadvantage of these tubes primarily applies to biologic therapeuticmolecules since the PK and immunogenicity assays used are typicallyimmunoassays that recognize the binding site (e.g., the idiotype, in thecase of a therapeutic antibody). If the therapeutic target is activatedex vivo, it can bind the biologic present in the plasma and thereby maskdetection in the PK and immunogenicity immunoassays. The proteaseinhibitors in the tubes can prevent the target activation. The use ofthe liquid formulation prevents hemolysis, which can interfere incertain laboratory assays.

Evacuated Blood Collection Tubes Containing Protease Inhibitor Cocktailsin Liquid Formulation

Evacuated blood collection tubes are commonly used in medical practicesfor collecting blood samples for various uses. The tubes describedherein may be non-glass tubes comprising a liquid formulation thatcomprises a mixture of protease inhibitors (a protease inhibitorcocktail). In some embodiments, the protease inhibitor cocktail maycomprise at least one serine protease inhibitor and at least onecysteine protease inhibitor. At least one serine protease inhibitor canbe a plasma kallikrein inhibitor. Such protease inhibitor cocktails maycomprise multiple (e.g., 2, 3, 4, or 5) serine protease inhibitors, atleast one of which can be a trypsin or human plasmin inhibitor.Preferably, the protease inhibitor cocktails described herein aresubstantially free of a protease inhibitor that is unstable in anaqueous solution, i.e., the activity of the protease inhibitor that isunstable in an aqueous solution is insubstantial as relative to thetotal inhibitory activity of the protease cocktail. In some instances,the amount of the protease inhibitor that is unstable in an aqueoussolution may be less than 5% (w/w) of the total protease inhibitors inthe cocktail, e.g., less than 2%, less than 1%, or less than 0.5%. Insome instances, the protease inhibitor cocktail is completely free of aprotease inhibitor that is unstable in an aqueous solution (e.g., anaqueous solution having a pH of 4-6). One example of protease inhibitorthat is not stable in an aqueous solution is PPACK II, also known asH-D-Phe-Phe-Arg-chloromethyl ketone.

Table 1 below lists exemplary serine protease inhibitors, cysteineprotease inhibitors, and trypsin protease inhibitors, which can be usedfor making the protease inhibitor cocktails described herein.

Categories Exemplary Inhibitors Serine Protease * BenzamidineInhibitors * 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride(AEBSF); * * Chymostatin; * Nalpha-Tosyl-Lys Chloromethyl Ketone(TLCK); * Tos-Phe-CH2Cl; N-p-Tosyl-L-phenylalanine chloromethyl ketone(TPCK) *1-({(6R,7S)-3-[(acetyloxy)methyl{-7-methoxy-5,5-dioxido-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-yl}carbonyl)-L-proline * Patamostatmesylate; *Gabexate mesylate; * Msaapvck (Meosuc-aapv-cmk;MeOSuc-Ala-Ala-Pro-Val-CMK) * Nafamostat mesylate; * Rosmarinic acid; *Purpurogallin; *2-(4-((1-Acetimidoyl-3-pyrrolidinyl)oxy)phenyl)-3-(7-amidino-2-naphthyl)propanoic acid hydrocloride pentahydrate *4-(4-Bromophenylsulfonylcarbamoyl)benzoyl-L-valyl-L-proline-1(RS)-(1-trifluoroacetyl-2-methylprolyl)amide *L-658758; CHEMBL446371; L658758 * Sivelestat; * Patamostat; * Cholesterol sulfate; * ElastaseInhibitor III; * Gabexate; * 4′,6-Diamidino-2-phenylindole; *4-aminobenzamidine; * 3,4-dichloroisocoumarin; * BivalirudinTrifluoroacetate * Pradaxa; * HIRUDIN; * Ximelagatran; * Lepirudin;Refludan; Hbw 023 * Bivalirudin; * Letaxaban; * Eribaxaban; * Dabigatranetexilate mesylate; * Apixaban; * Tanexaban; * Rivaroxaban; Xarelto;366789-02-8 * Plasma kallikrein inhibitors such as EPL-KAL2, DX-88,DX-2930, etc. The following examples are trypsin and/or human plasmininhibitors: * Soybean trypsin inhibitor *4-(2-aminoethyl)benzenesulfonylfluoride * 4-aminobenzamidine * alpha1-Antitrypsin * Aprotinin * Camostat * Eco protein (E coli) *inter-alpha-inhibitor * Nafamostat * NCO 650 * Ovomucin * SomatomedinB * Trypsin Inhibitor (Bowman-Birk Soybean) * Trypsin Inhibitor (KunitzSoybean) * Urinastatin Cysteine Protease * Geldanamycin; 30562-34-6;AKOS022185390 Inhibitor * Calpastatin; *L-Proline,N-[[2S,3S)-3-[(propylamino)carbonyl]-2-oxiranyl]carbonyl]-L-isoleucyl-; * Proteasome Inhibitor I; *(L-3-trans-(Propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline; *Calpain Inhibitor III; *[L-3-trans-(Propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline; * Omuralide; * (S)-MG132; * Lactacystin; *Z-Phe-ala-diazomethane; * Leupeptin; * 4-Hydroxynonenal; *trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane; * Loxistatin; *Clasto-lactacystinbeta-lactone; * L-Proline, * Z-FA-FMK; *N-acetylleucyl-leucyl-methioninal; * nitroaspirin; * Allnal; *Aloxistatin; * ethyl3-({4-methyl-1-[(3-methylbutyl)amino]-1-oxopentan-2-yl}carbamoyl)oxirane-2-carboxylate; * (+/−)4-HYDROXYNON-2-ENAL;

In some examples, the protease inhibitor cocktail for use in making theevacuated blood collection tubes comprises at least one serine proteaseinhibitor (e.g., 1, 2, or 3), which may include at least onetrypsin/plasmin inhibitor (e.g., 1, 2, or 3), and at least one cysteineprotease inhibitor (e.g., 1, 2, or 3). Such a protease inhibitorcocktail may comprise three serine protease inhibitors (e.g.,benzamidine, AEBSF, and a trypsin/plasmin inhibitor such as soybeantrypsin inhibition) and one cysteine protease inhibitor (e.g.,leupeptin).

In other examples, the protease inhibitor cocktail may comprise at leastone serine protease inhibitor (e.g., a plasma kallikrein inhibitor) andat least one cysteine protease inhibitor (e.g., leupeptin). The plasmakalikrein inhibitor may be EPI-KAL2 (Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp; SEQ ID NO: 1),which is a specific plasma kallikrein, recombinant protease inhibitorthat offers the ability for the tubes to contain a reagent that permitsdetection of activated plasma kallikrein using, e.g., immunoassays.

Any of the protease inhibitor cocktails may be dissolved in a suitablesolution to form a liquid formulation. The suitable solution may be anacid-citrate-dextrose solution, which may comprise trisodium citrate,citric acid, and dextrose. The solution may have a pH value of about4-6, 4-5, 4.5-5.0, or 4.2-4.7, e.g., 4.5. In some embodiments, thesolution has a pH value of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. Insome embodiments, the solution has a pH value of about 4.5. The liquidformulation may further comprise a cationic polymer such ashexadimethrine bromide molecule (Polybrene®), which can reduce contactsystem activation by interaction with negatively charged surfaces and achelating agent (e.g., EDTA), which can inhibit metalloproteases.

The concentration of each of the protease inhibitors in the cocktail maybe 5× or 10× higher than the final concentration of such an inhibitorfor use in inhibiting the corresponding protease, depending upon thedilution fold in practice. The final concentration of a specificcommercially protease inhibitor was known in the art and can be obtainedfrom manufacturer's protocol. In some examples, the concentration ofEPI-KAL2 may range from 5-15 μM (e.g., 5-10, 7 to 12 μM, or 10-15 μM).In some embodiments, the concentration of EPI-KAL2 is about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or about 15 μM. In some examples, theconcentration of leupeptin may range from 200-300 μM (e.g., 200-250,240-270, or 250-300 μM). In some embodiments, the concentration ofleupeptin is about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, orabout 300 μM. In some examples, the concentration of soybean trypsininhibitor may range from 1-3 mg/ml (e.g., 1-2 or 2-3 mg/ml). In someembodiments, the concentration of soybean trypsin inhibitor is about 1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, or about 3.0 mg/mL. In some examples, theconcentration of benzamidine can range from 80-120 mM (e.g., 80-100 or100-120 mM). In some embodiments, the concentration of benzamidine isabout 80, 85, 90, 95, 100, 105, 110, 115, or about 120 mM. In someexamples, the concentration of AEBSF may range from 10-30 mM (e.g.,10-20 or 20-30 mM). In some embodiments, the concentration of AEBSF isabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or about 30 mM.

When a peptide-based protease inhibitor (e.g., EPI-KAL2) is used, it maybe biotinylated following conventional methodology. For example, thepeptide inhibitor may be biotinylated as follows. Briefly, the peptideinhibitor can be dissolved in a suitable solution, such asphosphate-buffered saline (PBS). Freshly prepared Sulfo-NHS-LC-Biotincan be added to the peptide inhibitor solution and incubated on ice fora suitable period of time. Excess non-reacted and hydrolyzed biotin canbe removed using a spin-desalting column. The labeling of the peptideinhibitor can be confirmed by ELISA and the protein concentration can bedetermined, for example, by the Bradford assay.

Any of the liquid formulations described herein can be prepared byroutine methods, e.g., dissolving the proper components into a suitablesolution, and placed in evacuated blood collection tubes, whichpreferably is non-glass. The tubes may be stored at −20° C. and may bethawed on ice or at refrigerated temperatures (e.g., about 4° C.), suchas in a refrigerator within a suitable period of time prior to use.

Utilities of Evacuated Blood Collection Tubes Comprising ProteaseInhibitor Cocktails in Liquid Form

Any of the evacuated blood collection tubes described herein can be usedto collect blood samples from subjects for use in analyzing endogenousfeatures associated with the contact system, including, but not limitedto, the level of contact system activation, the serum level of a drugthat targets a component of the contact system, and/or theimmunogenicity of such a drug. To reduce ex vivo activation of thecontact system (e.g., by local trauma following vessel puncture by theneedle), the evacuated blood collection tubes described herein may notbe the first tube filled with blood when drawing blood from a subject.For example, the initial blood from the subject may be collected in afirst tube, which may be discarded, and then the evacuated bloodcollection tubes are used to collect the subsequent blood samples, whichcan be used for analysis. The first tube may be a regular bloodcollection tube used in routine practice.

After blood collection, the blood samples may be processed to produceplasma samples within a suitable period of time (e.g., not exceeding anhour). The plasma samples can be subject to further analysis to assessfeatures associated with the contact system of the subject from whom theinitial blood sample is obtained.

The blood samples may be collected from a subject in need of theanalysis as described herein. In some instances, the subject is a humanpatient, who may have, be suspected of having, or be at risk for adisease associated with the contact system. For example, the humanpatient may have a prior occurrence of HAE or may be at risk for HAE.The human patient may have type I or type II HAE, who either isdeficient in C1-INH or produces an atypical C1-INH. Alternatively, thehuman patient may have type III HAE, which is not connected with C1-INHdeficiency. In other examples, the human patient may have a prioroccurrence of idiopathic angioedema or may be at risk for idiopathicangioedema. Such a human patient may have been treated previously or bein the course of treatment with a drug that targets a component of thecontact system (e.g., pKal or FXIIa or high molecular weight kininogen).

i. Assessing Endogenous Level of Contact System Activation

In one aspect, the plasma samples noted herein can be analyzed to assessthe endogenous level of contact system activation of a subject, whichmaybe a human patient having, suspected of having, or at risk for adisease associated with the contact system (e.g., HAE or idiopathicangioedema). Such a human patient may be on a treatment of the disease,for example, a treatment involving a pKal inhibitor (e.g., an anti-pKalantibody). In other instances, such a human patient may be free of sucha treatment. Alternatively, the human subject may be a healthy subjecthaving no such diseases.

The level of contact system activation of the plasma sample can bedetermined by measuring one or more biomarkers indicative of contactsystem activation.

Plasma kallikrein (pKal) is the primary bradykinin-generating enzyme inthe circulation. The activation of pKal can occur via the contact systemor via Factor XIIa, both of which have been linked to disease pathologyassociated with hereditary angioedema (HAE). Plasma kallikreincirculates as an inactive zymogen called prekallikrein that is mostlybound to its substrate high molecular weight kininogen (HMWK). Inresponse to a stimulus, prekallikrein is cleaved to form active plasmakallikrein. This activation of kallikrein can be mediated, e.g., byFactor XIIa after activation of FXII to FXIIa, or by an effector of thecontact cascade. Approximately 75-90% of circulating prekallikrein isbound to HMWK through a non-active site interaction with domain 6 ofHMWK that will hydrolyze additional molecules of HMWK to generatecleaved HMWK and bradykinin. Active plasma kallikrein cleaves HMWK attwo sites, resulting in the release of Bradykinin, a key mediator ofpain, inflammation, edema and angiogenesis. The other cleavage product,cleaved kininogen, comprises to amino acid chains held together by adisulfide bond. Cugno et al., Blood (1997) 89:3213-3218.

Exemplary biomarkers that can be used to assess the level of contactsystem activation in a patient blood sample (thus determining whetherthe patient has an elevated level and/or activity of the contact system,e.g., an elevated level or activity of pKal), are provided in Table 2below:

TABLE 2 Contact System Biomarkers Basal Level in HAE Δ indicating orduring Biomarker Assay patient relative to normal acute edema attackPrekallikrein ELISA or enzyme Unchanged (~500 nM) or Decreased furtheractivity assay slightly decreased Active pKal ELISA or enzyme Unchangedor slightly Increased activity increased α2M-pKal complex ELISAElevated, if recent attack Increased C1INH-pKal ELISA Elevated, ifrecent attack Increased complex FXIIa ELISA or enzyme Unchanged orslightly Increased activity increased FXIa ELISA or enzyme Unchanged orslightly Increased activity increased Intact HMWK ELISA Unchanged orslightly Decreased decreased Cleaved HMWK ELISA Unchanged or slightlyIncreased increased Bradykinin ELISA or LC-MS Unchanged or slightlyIncreased metabolite increased

One or more of the biomarkers indicative of contact system activationmay be analyzed using convention methods. One particularly suitable typeof assay for detecting, either qualitatively, semi-quantitatively, orquantitatively, is immunoassays. An immunoassay is any assay in which atarget molecule (e.g., a biomarker molecule associated with contactsystem activation) is detected and/or quantified by using a bindingagent as described herein that specifically binds the target molecule.The binding agent may be an antibody, which can be a full-lengthantibody or an antigen-binding fragment thereof. The immunoassay may bea competitive or a non-competitive immunoassay, and may be a homogeneousor a heterogeneous immunoassay. For example, the immunoassay fordetecting a contact system biomarker may be an enzyme immunoassay (EIA),radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescentimmunoassay (CLIA), counting immunoassay (CIA), immunoenzymometric assay(IEMA), enzyme-linked immunosorbent assay (ELISA), a lateral flowimmunoassay, a sandwich immunoassay, an immuno-PCR assay, a proximityligation assay, a western blot assay, or an immunoprecipitation assay.Additional suitable immunoassays for detecting a biomarker providedherein will be apparent to those of skill in the art. It will beapparent to those of skill in the art that this disclosure is notlimited to immunoassays, however, and that detection assays that are notbased on an antibody or an antigen binding antibody fragment, such asmass spectrometry, are also useful for the detection and/orquantification of contact system biomarkers as provided herein.

The type of detection assay used for the detection and/or quantificationof a contact system biomarker such as those provided herein will dependon the particular situation in which the assay is to be used (e.g.,clinical or research applications), and on the kind and number ofbiomarkers to be detected, and on the kind and number of patient samplesto be run in parallel, to name a few parameters. For example, elevatedlevels of cleaved kininogen (2-chain kininogen) can be detected inplasma samples collected from HAE patients or healthy subjects during anacute HAE episode using a Western blot assay. While Western blot assaysallow for the simultaneous analysis of contact system biomarkers in aplurality of samples, they are limited in the number of biomarkers thatcan be assessed in parallel. Accordingly, in some embodiments, where aplurality of contact system biomarkers provided herein is analyzed in asingle sample, or in multiple samples, assays suitable for suchmultiplex analysis are preferred. Examples for such assays include,without limitation, peptide microarrays and lab-on-a-chip assays, whichhave been designed to offer a high throughput, multiplex-readyalternative to less scalable immunoassays such as Western blots.

In some examples, a plasma sample can be placed in a multi-wellmicroplate in the presence or absence of a pKal inhibitor and/or acontact system activator. The mixture can be incubated on ice in thepresence of a labeled peptide substrate of pKal for a suitable period oftime (e.g., 2 minutes), and corn trypsin inhibitor (CTI) can be added tothe mixture to stop the activation reaction. The mixture can be dilutedif necessary and the proteolytic activity can be determined by measuringthe level of a fluorescent peptide substrate. The results obtained formsuch an assay can be relied on to determine the endogenous level ofcontact system activation of the subject from whom the plasma sample isobtained. They also can be relied on to determine the inhibitoryactivity of the pKal inhibitor, if used.

ii. Assessing Endogenous Levels of Drugs Targeting the Contact System

Another aspect of the present disclosure pertains to the use of theevacuated blood collection tubes described herein for the determinationof the levels of a drug targeting a component of the contact system.Drug levels are required to evaluate pharmacokinetic parameters. Forexample, if the contact system is activated ex vivo in plasma samplescollected for the determination of amount of a plasma kallikreininhibitor (e.g., DX-2930) in plasma, then the excess activated plasmakallikrein could bind the drug and thereby mask its detection in anassay. Any of the evacuated blood collection tubes described herein canbe used to more accurately estimate drug levels, for example in a samplecollected from a subject.

To practice this method, plasma samples derived from a subject (e.g., ahuman patient) treated with a drug that targets a component of thecontact system (e.g., pKal) may be prepared from blood samples collectedin the evacuated blood collection tubes described herein following themethods also described herein. The level of the drug in the plasmssample can be measured following routine practice. In some instances,the drug level can be measured by an immunoassay, e.g., those describedherein.

iii. Assessing Immunogenicity of Drugs Targeting the Contact System

Another aspect of the present disclosure pertains to the use of theevacuated blood collection tubes for the determination of theimmunogenicity of a biologic inhibitor against a component of thecontact system (e.g., pKal). For example, it is common practice todevelop immunogenicity assays that are able to measure antibodiesagainst the drug (“ADAs”) in the presence of excess drug in the plasmaof the circulation. This requirement for an immunogenicity assay thatcan measure ADAs is certainly the case for therapeutic monoclonalantibodies, which can have multiple week half-lives and high drug levelsin the circulation. To overcome the interference due to excess drug inthe sample, techniques are performed to separate anti-drug antibodiesfrom the drug. Such anti-drug antibodies may be isolated by solid phaseextraction and acid dissociation (SPEAD), which involves incubation of abiotinylated form of the drug with the plasma sample for an extendedperiod of time (usually overnight) followed by isolation of thebiotinylated drug that may be bound to anti-drug antibodies using astreptavidin coated plate. The plate is then treated with acid torelease the anti-drug antibodies. The released antibodies may be coateddirectly on another assay plate for detection. In the absence ofprotease inhibitors in the collection tubes, the contact system canbecome activated ex vivo, leading to the production of active plasmakallikrein. After the acid releasing and re-coating steps noted above,both active pKal and anti-drug antibodies would bind to the surface ofthe plate. The anti-drug antibody is typically detected using a labeleddrug, which could also bind to active pKal, if present, resulting in afalse positive signal in the ADA assay.

The use of the evacuated blood collection tubes that comprise theprotease inhibitor cocktails described herein can prevent this falsepositive signal.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Preparation of Protease Inhibitor Cocktails that PreventContact System Activation

Protease inhibitor cocktails that prevented contact system activationwere developed. Evacuated plastic tubes were used for standardized andsimplified blood collection.

The following two protease inhibitor cocktails were utilized in a liquidformulation to prevent hydrolysis:

1) 10× Protease Inhibitor Cocktail A: SCAT169

-   -   Evacuated 5 mL total volume plastic tubes containing (0.5 ml):        100 mM benzamidine, 400 μg/mL polybrene, 2 mg/mL soybean trypsin        inhibitor, 20 mM EDTA, 263 μM leupeptin, and 20 mM AEBSF        (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)        dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67        mM citric acid, and 2% dextrose, pH 4.5.).

2) 10× Protease Inhibitor Cocktail B: SCAT153

-   -   Evacuated 5 ml total volume plastic tubes containing (0.5 ml):10        μM biotinylated EPI-KAL2, 400 μg/mL polybrene, 20 mM EDTA, and        263 μM leupeptin, dissolved in acid-citrate-dextrose (100 mM        trisodium citrate, 67 mM citric acid, and 2% dextrose, pH 4.5.).

Biotinylated EPI-KAL2 was included in the Protease Inhibitor Cocktail B(SCAT153). SCAT169 (Specialized Coagulation Assay Tubes, formulation169) and SCAT153 (Specialized Coagulation Assay Tubes, formulation 153)tubes were stored at 2-8° C.

Example 2: SCAT169 and SCAT153 Tubes Prevent Contact System Activationas Shown by 2-Chain Western Blot Assay

Plasma collected in either the SCAT169 or the SCAT153 tubes blockedcontact system activation induced by ex vivo addition of ellagic acid(FIG. 1), a well-known contact system activator, as measured by theconversion of 1-chain to 2-chain HMWK by Western blot analysis.

Observed ellagic acid-induced contact system activation was comparedbetween plasma samples collected in 3 different blood collection tubes:sodium citrate tubes (standard tubes used in clinical chemistry labs forcoagulation measurements), SCAT169 tubes, and SCAT153 tubes.

1-chain HWMK was essentially completely consumed in sampled activatedwith ellagic acid in sodium citrate tubes, and the appearance of 2-chainHMWK was detected.

In contrast, 1-chain HMWK was preserved in ellagic acid-activated plasmafrom SCAT169 and SCAT153 tubes.

These results provided evidence that the SCAT169 and SCAT153 tubes areeffective in preventing ex vivo contact system activation that can occurduring plasma sample collection and processing.

Example 3: SCAT169 and SCAT153 Protease Inhibitor Cocktails IncreasedPlasma Clotting Times

Plasma clotting times were measured in samples treated in 3 differentblood collection tubes: sodium citrate tubes, SCAT169 tubes, and SCAT153tubes, for three individual donor samples (Table 1).

As shown through prothrombin and activated partial thromboplastin,clotting time increased in samples with SCAT159 and SCAT153 as comparedto sodium citrate. The results are provided in Table 3 below.

TABLE 3 Effect of the Protease Inhibitors on Plasma Clotting TimesActivated Partial Prothrombin Time (s) Thromboplastin Time (s) SCAT SCATDONOR#: Citrate SCAT169 153 Citrate SCAT169 153 1 12.4 214.55 55.342.8 >240 >240 2 12.8 159.6 27.85 37.6 >240 >240 3 11.4 >300 87.134.6 >240 >240

Example 4: Use of Evacuated Blood Collection Tubes for AssessingEndogenous Levels of Contact System Activation in Human Subjects

Examination of plasma biomarkers of contact system activation ischallenging due to inadvertent activation during blood collection andprocessing. In this study, the utility of specialized blood collectiontubes (SCAT159 and SCAT153) in assessing levels of cleavedhigh-molecular-weight kininogen (cHMWK) in plasma from healthy subjectsand those with Types I/II hereditary angioedema (HAE), idiopathicangioedema or HAE with normal C1-INH (HAEnC1, also referred to asnC1-INH) was investigated as follows.

To avoid artificial activation of the contact pathway during bloodsampling, the study used standardized blood collection techniques andcustom tubes containing protease inhibitors. Blood samples werecollected from healthy subjects and the disease subjects noted above(during periods of disease quiescence and flare) to assess thepercentage of cHMWK using a Western blot assay. The blood samples wereplaced in SCAT159 or SCAT153 tubes (5 mL total volume, 0.5 mL 10×protease inhibitor cocktail) using a catheter with a butterfly-typeneedle system. The blood samples were then processed to produce plasmasamples within 1 hour after blood collection.

The SCAT tube plasma samples were analyzed by Simple Western (SBHD) andWestern blot (TGA) to determine the level of cleaved kininogen (2-chainkininogen) in the plasma samples following methods disclosed in, e.g.,WO 2015/061183.

Plasma was collected from the healthy subjects at clinical sites 1-5into plastic collection tubes containing various anti-coagulants,protease inhibitors, or protein stabilizers before being processed toplasma. Specifically, the types of tubes were K₂EDTA, sodium citrate,SCAT169 or P100 (BD Biosciences). As shown in FIG. 2, the tubes thatwere found to minimize ex vivo contact system activation containedprotease inhibitors and were either P100 tubes or SCAT169 tubes, whichwere 5 mL volume plastic evacuated blood collection tubes containing 0.5mL 10× concentrated mixture of 100 mM benzamidine, 400 μg/mL polybrene,2 mg/mL soybean trypsin inhibitor, 20 mM EDTA, 263 μM leupeptin, and 20mM AEBSF dissolved in acid-citrate-dextrose (100 mM trisodium citrate,67 mM citric acid, and 2% dextrose, pH 4.5). Collection methods usingtubes containing the protease inhibitor cocktails prevented elevation ofcHMWK when healthy volunteer plasma was assayed.

Results obtained from this study showed that, in healthy subjects,levels of cHMWK were stable (<5%) at room temperature (RT) for at least24 hours following blood collection into custom tubes, but the levels ofcHMWK were elevated (12%) in plasma samples obtained from a commercialvendor, demonstrating the importance of optimizing blood collectiontechniques when examining contact pathway activation. protease

The effects of kininogen cleavage in plasma collected into SCAT169 tubesfrom healthy subjects from two two different clinical sites (sites 4 and5) and from a commercial vendor (site 1) was also evaluated (FIG. 3).The commercial vendor collected blood samples directly into the SCAT 169tubes without an initial discard tube.

Kininogen cleavage was also evaluated in blood samples collected inSCAT169 tubes from subjects having types of angioedema (type I/II HAE,nC1-INH HAE, and idiopathic AE). Plasma was collected from healthysubjects or subjects having various types of angioedema in both thequiescent state (basal) and during an attack (attack) to measure theamount of cHMWK in various disease states. cHMWK percentages wereclearly elevated at baseline in subjects with HAE (n=21) compared tohealthy controls (n=26) but not in plasma from subjects with idiopathicangioedema (n=4) or HAEnC1 (n=5) (FIG. 4), suggesting limited plasmakallikrein activity, although not excluding a role of contact pathwayactivation during acute attacks in those disorders.

Plasma evaluated for detection of cHMWK is vulnerable to contact systemactivation stimulated by the collection methods and tubes. In sum, thisstudy provides a refined method for collection of plasma samples forevaluation of contact system activation, which prevents cleavage of HMWKex vivo.

Example 5: Use of Evacuated Blood Collection Tubes for AssessingEndogenous Levels of Contact System Activation in Human Subjects

The specialized blood collection tubes (SCAT159 and SCAT153) wereevaluated for utility in assessing levels of cleavedhigh-molecular-weight kininogen (cHMWK) in plasma from healthy subjectsand those with Types I/II hereditary angioedema (HAE).

Briefly, blood samples were collected from the healthy subjects and frompatients having HAE, during periods of disease quiescence (basal) andflare (attack). The percentage of 2-chain HMWK in the plasma wasdetected using a Western blot assay. Plasma samples were collected froma phase 1b multicenter, double-blind study of randomized patients withHAE type I/II who received 2 subcutaneous doses of anti-pKal antibody(DX-2930) on days 0 and 15 in close groups of 30, 100, 300, or 400 mg orplacebo. Blood samples were obtained prior to and after the anti-pKalantibody (DX-2930) on days 1, 8, 22, 64, 92, and 120.

As shown in FIG. 5A, the percentage of plasma 2-chain HMWK levels insamples from healthy subjects collected from three different clinicalsites (A, B, and C) varied with the collection method and type of tubeused. Samples using sodium citrate tubes had higher levels of 2-chainHMWK, relative to samples in SCAT169 tubes. Notably, samples collectedat clinical site C, which were collected directly into the SCAT169 tubesand did not use a discard tube prior to the tube containing the proteaseinhibitor cocktail, had higher levels of 2-chain HMWK as compared tosamples using a discard tube.

Similarly, as shown in FIG. 5B, the percentage of plasma 2-chain HMWKlevels in samples from HAE were higher in the sodium citrate tubes ascompared to plasma collected in SCAT169 tubes, likely due to exogenousactivation of the contact system associated with plasma collection andprocessing.

This study demonstrates the advantages of using the blood collectiontubes containing the protease inhibitor cocktails, as described herein,and provides a refined method for collection of plasma samples forevaluation of contact system activation, which prevents aberrant contactsystem activation (e.g., shown by the cleavage of HMWK) ex vivo.

Example 6: Use of Evacuated Blood Collection Tubes for Assessing PlasmaDrug Levels

Blood is drawn from HAE patients treated with DX-2930 and healthycontrols by routine practice and placed into collection tubes. Afterplacing the initial blood samples in one or multiple collection tubes, 5ml of the following blood sample is placed into SCAT159 or SCAT153tubes. The blood samples are then processed to produce plasma sampleswithin 1 hour after blood collection.

The amount of DX-2930 in the SCAT plasma samples are measured bystandard immunoassays, for example, ELISA. Briefly, a Fab version of ananti-idiotypic monoclonal antibody against DX-2930 is coated onto thesurface of a 96-well plate overnight and unbound anti-idiotypic Fabmolecules are removed by washing the plate multiple times. The SCATplasma samples are then added to the plate, which is incubated at roomtemperature for 2-3 hours. The plate is washed several times and abiotinylated IgG version of an anti-idiotypic monoclonal antibodyagainst DX-2930 is added to the plate followed by an incubation step anda wash step. Horseradish peroxidase conjugated streptavidin is thenadded to the plate. After a 30′ incubation, the plate is washed againand examined for a signal released by the dye. The intensity of thesignal corresponds to the amount of DX-2930 in the plasma sample.

Example 7: Use of Evacuated Blood Collection Tubes for Assessing DrugImmunogenicity

Plasma samples from HAE patients treated with DX-2930 are prepared fromblood samples collected in SCAT159 or SCAT153 tubes, as disclosed above.Anti-DX-2930 antibodies in the plasma sample are isolated by solid phaseextraction and acid dissociation (SPEAD).

Briefly, the plasma samples are incubated with biotinylated DX-2930 andincubated overnight. The mixture is then placed into a plate coated withstreptavidin to capture biotinylated DX-2930, which bind to anti-DX-2930antibodies in the plasma sample, if any. The anti-DX-2930 antibodies arethen released by acid treatment and coated directly on a Meso ScaleDiscovery (MSD) plate. Ruthenium-labeled DX-2930 is added to the MSDplate and an electrochemiluminescent signal is measured to detect thepresence of anti-DX-2930 antibodies.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the present disclosure described herein. The scope of thepresent disclosure is not intended to be limited to the abovedescription, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The present disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thepresent disclosure includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process.

Furthermore, the present disclosure encompasses all variations,combinations, and permutations in which one or more limitations,elements, clauses, and descriptive terms from one or more of the listedclaims is introduced into another claim. For example, any claim that isdependent on another claim can be modified to include one or morelimitations found in any other claim that is dependent on the same baseclaim. Where elements are presented as lists, e.g., in Markush groupformat, each subgroup of the elements is also disclosed, and anyelement(s) can be removed from the group. It should it be understoodthat, in general, where the present disclosure, or aspects of thepresent disclosure, is/are referred to as comprising particular elementsand/or features, certain embodiments of the present disclosure oraspects of the present disclosure consist, or consist essentially of,such elements and/or features. For purposes of simplicity, thoseembodiments have not been specifically set forth in haec verba herein.It is also noted that the terms “comprising” and “containing” areintended to be open and permits the inclusion of additional elements orsteps. Where ranges are given, endpoints are included. Furthermore,unless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the present disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the present disclosure can be excluded from anyclaim, for any reason, whether or not related to the existence of priorart.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present disclosure, as defined in the following claims.

What is claimed is:
 1. A method for assessing the endogenous level ofcontact system activation in a subject, the method comprising: (i)collecting blood from a subject to an evacuated blood collection tube;(ii) processing the blood to produce a plasma sample; and (iii)measuring the level of contact system activation in the plasma sample,wherein the evacuated blood collection tube comprises a liquidformulation that comprises a mixture of protease inhibitors, polybrene,and EDTA, wherein the mixture of protease inhibitors comprisesbenzamidine, soybean trypsin inhibitor, leupeptin, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).
 2. A method forassessing the level of a drug targeting the contact system in a subject,the method comprising: (i) collecting blood from a subject to anevacuated blood collection tube; wherein the subject has beenadministered a drug that targets a component of the contact system; (ii)processing the blood to produce a plasma sample; and (iii) measuring thelevel of the drug in the plasma sample, wherein the evacuated bloodcollection tube comprises a liquid formulation that comprises a mixtureof protease inhibitors, polybrene, and EDTA, wherein the mixture ofprotease inhibitors comprises benzamidine, soybean trypsin inhibitor,leupeptin, and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride(AEBSF).
 3. A method for assessing immunogenicity of a drug targetingthe contact system, the method comprising: (i) collecting blood from asubject to an evacuated blood collection tube; wherein the subject hasbeen administered a drug that targets a component of the contact system;(ii) processing the blood to produce a plasma sample; and (iii)measuring the level of antibodies that bind to the drug in the plasmasample, wherein the evacuated blood collection tube comprises a liquidformulation that comprises a mixture of protease inhibitors, polybrene,and EDTA, wherein the mixture of protease inhibitors comprisesbenzamidine, soybean trypsin inhibitor, leupeptin, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).
 4. The method of claim1, wherein the tube is a non-glass tube.
 5. The method of claim 1,wherein the liquid formulation comprises 80-120 mM benzamidine, 1-3mg/ml soybean trypsin inhibitor, 200-300 μM leupeptin, and 10-30 mMAEBSF.
 6. The method of claim 1, wherein the liquid formulationcomprises 100 mM benzamidine, 2 mg/ml soybean trypsin inhibitor, 263 μMleupeptin, and 20 mM AEBSF.
 7. The method of claim 1, wherein thepolybrene is at a concentration of 400 μg/mL and the EDTA is at aconcentration of 20 mM.
 8. The method of claim 1, wherein the liquidformulation further comprises trisodium citrate, citric acid, anddextrose.
 9. The method of claim 8, the trisodium citrate is at aconcentration of 100 mM, the citric acid is at a concentration of 67 mM,and the dextrose is 2%.
 10. The method of claim 1, wherein the liquidformulation has a pH of 4-6.
 11. The method of claim 10, wherein theliquid formulation has a pH of 4.5.
 12. The method of claim 1, whereinthe mixture of protease inhibitors consists of benzamidine, soybeantrypsin inhibitor, leupeptin, and AEBSF.
 13. The method of claim 1,wherein in step (i), the evacuated blood collection tube is not thefirst tube filled with the blood from the subject; and/or step (ii) isperformed within one hour after step (i).
 14. The method of claim 1,wherein the subject is a human subject having a disease associated withthe contact system.
 15. The method of claim 14, wherein the disease ishereditary angioedema (HAE) or idiopathic angioedema.
 16. The method ofclaim 14, wherein the human subject is treated with a drug targeting acomponent of the contact system, optionally plasma kallikrein (pKal).17. The method of claim 16, wherein the drug inhibits active plasmakallikrein.
 18. The method of claim 1, wherein step (iii) is performedby measuring the level of one or more biomarkers indicative of contactsystem activation.
 19. The method of claim 18, wherein the one or morebiomarkers are selected from the group consisting of prekallikrein,active plasma kallikrein (pKal), α2M-pKal complex, active Factor XII,active Factor XI, high molecular weight kininogen (HMWK), and abradykinin metabolite.
 20. The method of claim 3, wherein the methodfurther comprises, prior to step (iii), isolating antibodies that bindto the drug from the plasma sample.